Combined chemo-immunotherapy with liposomal drugs and cytokines

ABSTRACT

A method of antitumor therapy is described in which administration of a chemotherapeutic drug, encapsulated in liposomes, is supplemented by administration of an immunostimulating cytokine. The cytokine is preferably also encapsulated in liposomes. In tumor models for lung and colon carcinomas, this method produced a significantly greater therapeutic effect, as evidenced by survival rate and tumor size, than a combination of the effects produced by the free or liposome-encapsulated components administered individually.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/IL98/00586, filed Dec. 1, 1998, whichclaims priority under 35 USC 119(e) to U.S. Provisional application No.60/067,697 filed Dec. 4, 1997.

FIELD OF THE INVENTION

The present invention relates to a method and composition for antitumortherapy, and more particularly to combination therapy using achemotherapeutic drug and an immunostimulating cytokine. Sequentialadministration of these two components, both encapsulated in liposomes,is shown to have a significant antitumor effect as compared toadministration of the individual components, in free form or inliposomes.

References

Adler, A. et al., Cancer Biotherapy 10:293-306 (1995).

Curran, D. P. et al., Angew. Chem. Intl. Ed. Eng. 34(23/24):2683-4(1996).

Gabizon, A. et al., Adv. Drug Delivery Reviews 24(2-3):337-344 (1997).

Kedar, E. et al., J. Immunotherapy 16:47-59 (1994).

Lasic, D. and Martin F., Eds., STEALTH LIPOSOMES CRC Press, Boca Raton,Fla. (1995).

Papahadjopoulos, D. et al., Proc. Natl. Acad. Sci. USA 88:11460-11464(1991).

Sears, B. D., U.S. Pat. No. 4,426,330 (1984).

Sears, B. D., U.S. Pat. No. 4,534,899 (1985a).

Szoka, F., Jr. et al., U.S. Pat. No. 4,235,871 (1980b).

Szoka, F., Jr. et al., Ann. Re. Biophys. Bioeng. 9:467 (1980).

Tirosh, O. et al., J. Chem. Soc. Perk. Trans. II 2:383-389 (1997).

Woodle, M. C. et al., U.S. Pat. No. 5,013,556 (1991).

BACKGROUND OF THE INVENTION

Despite prolific research in the area of cancer chemotherapy, suchtreatment remains far from satisfactory. The inability ofchemotherapeutic drugs to reach the tumor site, intrinsic and acquiredcross-resistance to multiple chemotherapeutic agents, and, especially,the high toxicity of many of these agents all contribute to treatmentfailures.

The use of immunostimulating cytokines, such as IL-2 and interferon-α,has proven to be effective in treatment of a proportion of patients withmalignancies such as melanoma and renal cell carcinoma, both alone andin combination with other therapeutic agents. However, major problemslimit their wide clinical use, including rapid plasma clearance,biodistribution to nonrelevant tissues, and high toxicity. Furthermore,their efficacy has been low in treatment of the most common tumors, e.g.colorectal, mammary, prostate, and lung carcinomas.

SUMMARY OF THE INVENTION

The present invention includes, in one aspect, a method of antitumortherapy, which comprises administering to a subject in need of suchtreatment, a therapeutically effective amount of a combination of achemotherapeutic drug and an immunostimulating cytokine, bothencapsulated in liposomes. In another aspect, the invention provides acomposition for use in antitumor therapy, which comprises such acombination of a chemotherapeutic drug and an immunostimulatingcytokine, both encapsulated in liposomes. Administration of thecombination produces a greater therapeutic effect than a combination ofthe effects produced by the liposome-encapsulated componentsadministered individually.

The invention also includes a method of antitumor therapy in which achemotherapeutic drug, encapsulated in liposomes, is administered incombination with a cytokine, which may or may not be encapsulated inliposomes. In this method, the drug is encapsulated in liposomes whichcontain about 1-10 mole percent of a lipid having a polar head groupderivatized with a polyethylene glycol (PEG) chain which has a molecularweight of between 750 and 10,000 daltons. The therapeutic effect of thiscombination is greater than a combination of the effects produced by theliposome-encapsulated drug and the cytokine administered individually.

In all cases, administration of the cytokine preferably followsadministration of the liposome-encapsulated drug.

The chemotherapeutic drug is preferably selected from cis-platin, achemotherapeutic anthraquinone, and a topoisomerase I inhibitor, such ascamptothecin or a camptothecin analog. More preferably, the drug isadriamycin (doxorubicin), in which case the liposome-encapsulated formof the drug is preferably DOXIL®, a polyethylene glycol-coated liposomaldoxorubicin.

The immunostimulating cytokine is preferably selected from the groupconsisting of interleukin-2 (IL-2), IL-12, IL-15, IL-18, IFN-γ, IFN-α,IFN-β, TNF-α, G-CSF, and GM-CSF. More preferably, the cytokine is IL-2.

The encapsulating liposomes employed in the composition and methodpreferably contain at least one lipid selected from dimyristoylphosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG),1,2-distearoyl-3-trimethylammonium propane (DSTAP), phosphatidylcholine, phosphatidyl ethanolamine, and cholesterol.

The liposomes may be small unilamellar vesicles (SUV), defined as havinga mean diameter of approximately 20 to 100 nm, or large unilamellarvesicles (LUV), defined as having a mean diameter of approximately 100to 200 nm. Such liposomes preferably contain about 1-10 mole percent ofa lipid having a polar head group derivatized with a polyethylene glycol(PEG) chain which has a molecular weight of between 750 and 10,000daltons.

Alternatively, the liposomes may be large multilamellar vesicles (MLV)having a mean diameter of approximately 250 to 2000 nm. The MLV may alsocontain a PEG-derivatized lipid as described above.

In preferred embodiments, the chemotherapeutic drug is encapsulated invesicles having a mean diameter of approximately 50 to 120 nm, andcontaining about 1-10 mole percent of a lipid having a polar head groupderivatized with a polyethylene glycol (PEG) chain as described above.In another preferred embodiment, the cytokine is encapsulated inliposomes containing dimyristoyl phosphatidyl choline (DMPC) plus 0 to50 mole percent of at least one lipid selected from dimyristoylphosphatidyl glycerol (DMPG) and 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP).

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the survival rate of BALB/c mice injected intraperitoneallywith 5×10⁵ M109 tumor cells (lung adenocarcinoma) and subsequentlytreated with free adriamycin or DOXIL®, respectively, alone or incombination with intraperitoneal IL-2 in DMPC/DMPG MLV liposomes, orwith liposomal IL-2 alone; and

FIG. 2 shows the survival rate of BALB/c mice injected intravenouslywith 5×10⁵ M109 tumor cells and subsequently treated with DOXIL® (at day7), alone or in combination with intravenous IL-2 in STEALTH® PEGylatedSUV liposomes (at days 11, 14, and 17), or with liposomal IL-2 alone (atdays 11, 14, and 17).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below have the following meanings unless indicated otherwise.

“Vesicle-forming lipids” refers to amphipathic lipids which havehydrophobic and polar head group moieties, and which (a) can formbilayer vesicles in water, as exemplified by phospholipids, or (b) canbe stably incorporated into lipid bilayers, with the hydrophobic moietyin contact with the interior, hydrophobic region of the bilayermembrane, and the polar head group moiety oriented toward the exterior,polar surface of the membrane.

The vesicle-forming lipids of this type typically include one or twohydrophobic acyl hydrocarbon chains or a steroid group, and may containa chemically reactive group, such as an amine, acid, ester, aldehyde oralcohol, at the polar head group. Included in this class are thephospholipids, where the two hydrocarbon chains are typically betweenabout 14-22 carbon atoms in length, and have varying degrees ofunsaturation. Representative examples are phosphatidyl choline (PC),phosphatidyl ethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SM), negatively charged lipids such asdimyristoyl phosphatidyl glycerol (DMPG), and positively charged lipidssuch as 1,2-distearoyl-3-trimethylammonium propane (DSTAP). Theliposomes may also contain sterols, such as cholesterol, which do notform liposomes themselves but can be incorporated into, and maystabilize, liposomes containing lipids such as those described above.

A “Cetus unit” (CU) is equal to six International Units (IU) ofImmunological Activity, the international reference standard of abiological preparation of interleukin-2 (IL-2). The term “unit” usedherein in reference to cytokine levels refers to Cetus units.

II. Liposomal Compositions

A. Lipid Components

Various vesicle-forming lipids, as defined above, may be used in thepresent liposomal compositions, according to methods well known in theart. Preferred lipids for the current invention allow long-term storageof the liposome-entrapped agents and effective release of thesecomponents upon administration. Representative lipids include, but arenot limited to, dimyristoyl phosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), cholesterol, egg phosphatidylcholine (eggPC), phosphatidyl ethanolamine (PE), distearoyl phosphatidylethanol-amine (DSPE), phosphatidyl inositol (PI),1,2-distearoyl-3-trimethylammonium propane (DSTAP),1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), and combinationsthereof.

The vesicle-forming lipids, preferably those making up SUV's, maycontain about 1-10 mole percent of a lipid having a polar head group,typically a phosphate containing head group, derivatized with apolyethylene glycol (PEG) chain which has a molecular weight of between750 and 10,000 daltons. The rate of clearance of liposomes fromcirculation is typically reduced by employing such PEG-derivatized, or“PEGylated”, lipids. PEG coating is believed to inhibit nonspecificadsorption of serum proteins, thereby preventing nonspecific recognitionof liposomes by macrophages (Papahadjopoulos, et al., 1991). Anotheradvantage of these long-circulating liposomes is their goodextravasation capacity and high accumulation in tumors (Lasic andMartin, 1995; Gabizon, et al., 1997). They are also referred to assterically stabilized liposomes, SSL, or STEALTH® liposomes.

The preparation of such lipids is described in, for example, Woodle, etal., 1991; Sears (1984, 1985); Tirosh et al. (1997) or copending andco-owned application having U.S. Ser. No. 08/570,440. The PEG chain maybe linked directly to the phosphatidic acid head group of aphospholipid. Various other linkages are possible; for example, lipidscontaining a phosphatidyl ethanolamine (PE) or other amino head groupmay be conveniently coupled to activated PEG chains via reaction withbrominated PEG. PEG-modified lipids are also commercially available,e.g. from Sequus Corporation, Menlo Park, Calif.

B. Preparation of Liposomes and Liposomal Compositions

Liposomes may be prepared by a variety of techniques, such as thosedetailed in Szoka et al. (1980b). To form multilamellar vesicles(MLV's), a mixture of vesicle-forming lipids dissolved in a suitablesolvent is evaporated in a vessel to form a thin film, which is thenhydrated by an aqueous median to form MLV's, typically with sizesbetween about 0.1 to 10 microns. Tert-butanol is a preferred solvent forthe process. The MLV's may then be downsized to a desired size range byextruding the aqueous suspension through a polycarbonate membrane havinga selected uniform pore size, typically 0.05 to 1.0 microns.

Preparations of MLV's or REV's (described below) may be treated, e.g. byextrusion, sonication or high pressure homogenization, to produceunilamellar vesicles. Small unilamellar vesicles (SUV's) arecharacterized by sizes in the 30-100 nm range, while large unilamellarvesicles (LUV's) are defined as those having mean diameters of about100-200 nm. SUV's may also be formed directly by high pressurehomogenization of an aqueous dispersion of lipids.

Various methods are available for encapsulating other agents inliposomes. Preparation of SSL-encapsulated IL-2 is described in Kedar etal. (1994). In this procedure, generally, the lipid components,including a PEG-substituted lipid, are dissolved in t-butanol. Thesolution is sonicated, and IL-2 is added with further sonication. Themixture is lyophilized and rehydrated, forming MLV's, which can then bedownsized by high pressure homogenization or by successive extrusionthrough polycarbonate filters. These downsizing methods gave vesicleshaving diameters of 50-80 nm and about 200 nm, respectively. Theprocedure achieved approximately 80-90% encapsulation of the IL-2.

In the reverse phase evaporation method (Szoka, et al., 1980a) anonaqueous solution of vesicle-forming lipids is dispersed with asmaller volume of an aqueous medium to form a water-in-oil emulsion. Theagent to be incorporated is included either in the lipid solution, inthe case of a lipophilic agent, or in the aqueous medium, in the case ofa water-soluble agent. After removal of the lipid solvent, the resultinggel is converted to liposomes. These reverse phase evaporation vesicles(REVs) have typical average sizes between about 0.2-4 microns and arepredominantly oligolamellar, that is, containing one or a few lipidbilayer shells. The REVs may be sized by extrusion, if desired, to giveoligolamellar vesicles having a maximum selected size between about 0.05to 1.5 microns.

Other methods for adding additional components to liposomal compositionsinclude colyo-philization with other components and redispersion of theresulting solid to form MLV's. In a method described by Adler, et al.(1995), an aqueous solution of the agent to be encapsulated is added toa t-butanol solution of lipids. The mixture is sonicated andlyophilized, and the resulting powder is rehydrated.

Liposome compositions containing an entrapped agent may be treated afterfinal sizing, if necessary, to remove free (non-entrapped) agent.Conventional separation techniques, such as centrifugation,diafiltration, and molecular-sieve chromatography are suitable for thispurpose. The composition may also be sterilized by filtration through aconventional 0.22 or 0.45 micron depth filter.

To form the compositions of the current invention, the concentration ofdrug and/or cytokine in the liposomes is preferably effective to give aprotein/lipid weight ratio between about 1:100 and 1:1000.

Stabilizers may also be added to the liposomal compositions. Forexample, addition of a metal chelator such as Desferal™ ordiethylenetriamine pentaacetic acid (DTPA) to the lyophilization medium,at a concentration of 100 μM, has been shown to reduce activity loss ofentrapped IL-2 during liposome preparation and storage at 4° C.Antioxidants such as BHT or Vitamin E may also be included.

For long term storage, the compositions may be stored as the drylyophilized powder, which is stable for at least a year at 4° C., andhydrated to form an aqueous suspension before use.

III. Combined Chemotherapy/Cytokine Therapy

A. Formulations

Cytokines useful for enhancing antitumor activity of chemotherapeuticdrugs include IL-2, IL-12, IL-15, IL-18, IFN-γ, IFN-α, IFN-β, TNF-α,G-CSF, and GM-CSF. A preferred cytokine for the present invention isIL-2 (interleukin 2), which acts as a growth and maturation factor forT-lymphocytes.

A variety of liposomal formulations may be used for encapsulation of thecytokine. These include MLV, LUV or SUV, as defined above, as well asOLV (oligolamellar vesicles) and MVV (multivesicular vesicles), composedof vesicle-forming lipids such as those described above. Combinations oflipids are generally most effective (see, for example, Kedar et al.,1994). One preferred type of formulation employs SUV or LUV, having amean diameter of approximately 50 to 120 nm, containing about 1-10 molepercent of a lipid having a polar head group derivatized with apolyethylene glycol (PEG) chain (also referred to as a PEGylated lipid).Formulation A below is one example. Other preferred formulations employdimyristoyl phosphatidyl choline (DMPC) and, optionally, up to 50 molepercent of at least one lipid selected from dimyristoyl phosphatidylglycerol (DMPG) and 1,2-distearoyl-3-trimethylammonium propane (DSTAP).In these formulations, the proportion of DMPG and/or DSTAP is morepreferably 5-25 mole percent. Formulation B below is one example. In allcases, small quantities (up to about one mole percent) of stabilizerssuch as tocopherol or Desferal™ may be included.

For the experiments described below, liposomal IL-2 was prepared in twoformulations, using IL-2 obtained from Chiron Corporation (Emeryville,Calif.), according to known methods such as those described above.Formulation A employed sterically stabilized (SSL) small unilamellarvesicles (SUV) composed of ²⁰⁰⁰PEG-DSPE (N-carbamyl-(polyethylene glycolmethyl ether)-1,2-distearoyl-sn-glycero-3-phosphoethanolaminetriethylammonium salt, provided by Sequus Corporation), egg phosphatidylcholine, and cholesterol in a molar ratio of about 5:55:40. The vesicleswere about 50-70 nm in diameter. Encapsulation efficiency of IL-2 wasgreater than 80%, based on an in vitro IL-2 bioassay (i.e., >80% of theinitial amount of added IL-2 became encapsulated in liposomes).

Formulation B employed multilamellar vesicles (MLV) composed ofDMPC-DMPG (dimyristoyl phosphatidyl choline—dimyristoyl phosphatidylglycerol) in a 9:1 molar ratio. The vesicles were approximately 500-1500nm in size, and the encapsulation efficiency was approximately >90%.This high efficiency of encapsulation was achieved at a lipid:IL-2 ratio(wt:wt) of 1000:1 for DMPC alone, and 100:1 for DMPC containing DMPG orDSTAP.

The chemotherapeutic drug is preferably encapsulated in liposomes havingabout 1-10 mole percent of a PEGylated lipid, as described above. Forexample, DOXIL®, a stable formulation of adriamycin in STEALTH®liposomes, is available from SEQUUS Pharmaceuticals, Inc. (Menlo Park,Calif.). Free adriamycin is available, e.g., from Cetus Oncology Corp.(Emeryville, Calif.) as a formulation of doxorubicin hydrochloride andlactose.

Other chemotherapeutic drugs which are also preferred for the presentmethod include other arthraquinones, such as epirubicin, daunorubicin,and mitoxanthrone, and cis-platin. Also contemplated are topoisomerase Iinhibitors such as camptothecin and its analogs, e.g. topotecan andirinotecan, also designated CPT-11. Camptothecin is isolated from thestem wood of the Chinese tree Camptotheca aciminata; preparation of theabove noted analogs has been described by, e.g., Curran et al. (1996).

B. Liposomal Adriamycin—Liposomal IL-2

The effect of adriamycin, used alone or in combination withinterleukin-2 (IL-2), where each component was in free orliposome-encapsulated form, on the survival rate of BALB/c mice infectedwith tumor cells, was tested as described below.

B1. Lung Adenocarcinoma Model: IL-2 in MLV. Six groups of BALB/c micewere injected intraperitoneally with 5×10⁵ M109 tumor cells (day 0).Free adriamycin or DOXIL®, respectively, were administered intravenouslyon day 7 at a dose of 8 mg/kg, and intraperitoneal cytokine treatmentwas initiated 3 days later. Liposomal IL-2 (formulation B; MLV DMPC/DMPG(9:1 mole ratio) liposomes containing IL-2) was given once daily (50,000CU/mouse) on days 10, 13 and 16. Control groups received no treatment orreceived the IL-2 treatment alone.

Each group, consisting of 8-9 mice, was inspected for survival up to 100days after tumor inoculation. Table I shows the number of survivors atthe end of the experiment and the median survival time obtained; FIG. 1shows the survival curves for all groups.

TABLE I NUMBER OF MEDIAN SURVIVING SURVIVAL GROUP TREATMENT MICE/TOTAL(DAYS) 1 Control 2/8 54 2 ADR 0/8 42 3 ADR + MLV-IL-2 5/8 >100 4 DOXIL ®5/8 >100 5 DOXIL ® + MLV-IL-2 8/8 >100 6 MLV-IL-2 1/9 21

As Table I shows, adriamycin (ADR) in combination with MLV-IL-2(liposomal IL-2, formulation B) was much more effective than eitheradriamycin alone or liposomal IL-2 alone, both of which showed lowersurvival rates than the control. When liposomal adriamycin (DOXIL®) wasadministered alone, or when non-liposomal adriamycin was combined withliposomal IL-2, five of eight mice survived for the duration of thetest.

The best result, i.e. survival of all subjects for 100 days or more, wasobserved for the combination of liposomal ADR (DOXIL®) with liposomalIL-2. In terms of number of surviving subjects, the effect of thecombination treatment was greater than a combination of the effects ofthe individual treatments.

B2. Metastatic Lung Adenocarcinoma Model: IL-2 in MLV (Formulation B)and PEG-Derivatized SUV (SSL). In this experiment, BALB/c mice wereinjected intravenously with 5×10⁵ M109 tumor cells (day 0). Freeadriamycin or DOXIL ®, respectively, were administered intravenously onday 7 (8 mg/kg), followed 3 days later by intravenous cytokinetreatment. Liposomal IL-2 (Formulation A; PEGylated SUV containing IL-2)was given once daily (50,000 CU/mouse) on days 11, 14 and 17. Controlgroups received no treatment or received the IL-2 treatment alone.

Each group, consisting of 8-9 mice, was inspected for survival up to 100days after tumor inoculation. Results are shown in Table II and FIG. 2.

TABLE II NUMBER OF MEDIAN SURVIVING SURVIVAL GROUP TREATMENT MICE/TOTAL(DAYS) 1 Control 0/8 43 2 ADR 2/8 56 3 DOXIL ® 1/8 66 4 SSL-IL-2 0/8 415 DOXIL ® + SSL-IL-2 7/9 >100

As a comparison of groups 3-5 shows, the combined treatment with DOXIL®and liposomal IL-2 was significantly more effective than treatment witheither liposomal component alone, particularly in terms of the number ofsubjects surviving for the duration of the test, i.e. 100 days or more(7 out of 9 compared to 0-1 out of 8). In this aspect, the combinedtreatment was significantly more effective than a combination of theeffects derived from the individual therapies.

In a second, more extensive study, nine groups of BALB/c mice wereinjected intraperitoneally with 5×10⁵ M109 tumor cells. Free adriamycinor DOXIL® (8 mg/kg) were administered intraperitoneally 7 days later,followed 3 days later by intravenous cytokine treatment. The cytokine,given once daily (50,000 CU/mouse) on days 10, 13 and 16, consisted offree IL-2, IL-2 in Formulation A (Stealth® PEGylated SUV), or IL-2 inFormulation B (9:1 molar DMPC/DMPG MLV).

Each group, consisting of 8 mice, and an untreated control group of 11mice, were inspected for survival up to 120 days after tumorinoculation. Results are shown in Table III.

TABLE III MEDIAN NUMBER OF TUMOR SURVIVAL GROUP TREATMENT FREEMICE/TOTAL (DAYS) 1 Control 3/11 69 2 ADR 4/8  100 3 ADR/free IL-2 3/8 53 4 ADR/MLV-IL-2 4/8  92 5 ADR/SSL-IL-2 3/8  72 6 DOXIL ® 4/8  102 7DOXIL ®/free IL-2 5/8  >120 8 DOXIL ®/MLV-IL-2 7/8  >120 9DOXIL ®/SSL-IL-2 5/8  >120

In this study, administration of free ADR and IL-2 showed little or nobenefit over free ADR alone (groups 2-5). However, combinations ofeither free or liposomal IL-2 with the chemotherapeutic drug inliposomes (DOXIL®) showed clear benefits over administration of the drugalone (groups 6-9). Overall, the groups (8 and 9) treated with acombination of both components in liposomes showed superior results.Group 8, in particular, showed a high survival rate and almost acomplete absence of tumors.

B3. Subcutaneous colon carcinoma model: IL-2 in MLV. In this test, 7groups of BALB/c mice were injected in the footpad with 10⁵ C26 coloncarcinoma cells. Seven days later, 8 mg/kg free or liposomal adriamycinwas administered i.v. Free or liposomal IL-2, as shown in Table IV, wasadministered i.p. according to the schedule described above. Results areshown in Table IV.

TABLE IV NUMBER OF NUMBER OF TUMOR FREE TUMOR FREE GROUP TREATMENT MICE,DAY 30 MICE, DAY 65 1 Control 0/7 0/7 2 ADR 0/7 0/7 3 ADR/free IL-2 0/70/7 4 ADR/MLV-IL-2 1/8 0/8 5 DOXIL ® 4/8 102 6 DOXIL ®/free IL-2 3/8 2/87 DOXIL ®/MLV-IL-2 6/8 4/8

As the data shows, administration of liposomal drug alone was somewhatbeneficial, but only the group receiving the combined liposomaltreatment showed significant recovery from tumors. In this group (group7), it was also observed that the tumors were significantly smaller thanin the other groups.

IV. Administration

For use in humans, a therapeutically effective dose of the compositiontypically corresponds to 20-100 mg adriamycin/m² of body surface. ForIL-2, a preferred dose corresponds to 50,000-500,000 CU per square meterof body surface. Administration may be by intraperitoneal (ip),subcutaneous (sc), intravenous (iv), intraarterial (ia), orintramuscular (im) injection. Liposomes in the form of largemultilamellar vesicles (MLV's) are preferred for intraperitoneal,subcutaneous or intramuscular administration, while SUV's are preferredfor intravenous as well as intramuscular administration.

As shown above, administration of liposome-encapsulated chemotherapeuticdrug is followed by administration of the liposome-encapsulatedcytokine. While specific time intervals and courses of treatment havebeen shown in the examples above, it is understood that dosages, timeintervals between courses, and the number of courses of treatment, forboth drug and cytokine, may be varied depending on the extent ofsymptoms and the condition of the patient.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications may be made without departing from the invention.

What is claimed is:
 1. A method for antitumor therapy, comprisingadministering to a subject in need of such treatment, a therapeuticallyeffective amount of a non-encapsulated chemotherapy drug and animmunostimulating cytokine, wherein the cytokine is encapsulated inmultilamellar liposomes (MLV), said treatment being characterized inthat the subject is administered on non-consecutive days with two ormore DOSES of said liposome encapsulated cytokine, a first dose beingadministered at least 3 days following administration of saidchemotherapeutic drug, wherein the time between administrations is suchthat the therapeutic effect of the combined administrations is greaterthan the sum of the therapeutic effects produced by administration ofsaid chemotherapeutic drug alone and by administration of saidimmunostimulating cytokine alone.
 2. The method of claim 1, wherein saidcytokine is selected from the group consisting of interleukin-2 (IL-2),IL-12, IL-15, IL-18, INF-γ, INF-α, INF-β, G-CSF, and GM-CSF.
 3. Themethod of claim 2, wherein said cytokine is IL-2.
 4. The method of claim1, wherein the liposomes comprise at least one lipid selected from thegroup consisting of dimyristoyl phosphatidyl choline (DMPC), dimyristoylphosphatidyl glycerol (DMPG), 1,2-distearoyl-3-trimethylammonium propane(DSTAP), phosphatidyl choline, phosphatidyl ethanolamine andcholesterol.
 5. The method of claim 4, wherein said cytokine isencapsulated in liposomes comprising (1) DMPC and (2) at least oneadditional lipid selected from the group consisting of dimyristoylphosphatidyl glycerol (DMPG), and 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP), said at least one additional lipid being in an amountof up to 50%.
 6. The method of claim 5, wherein said liposome iscomposed of DMPC and DMPG.
 7. The method of claim 6, wherein theliposome comprise DMPC and DMPG in a molar ratio of about 9:1.
 8. Themethod of claim 1, wherein said chemotherapeutic drug is selected fromthe group consisting of a chemotherapeutic anthraquinone, cisplatin, anda topoisomerase I inhibitor.
 9. The method of claim 8, wherein saidchemotherapeutic drug is doxorubicin (adriamycin).
 10. A method forantitumor therapy, comprising administering to a subject in need of suchtreatment, a therapeutically effective amount of a chemotherapeutic drugencapsulated in liposomes and an immunostimulating cytokine encapsulatedin MLV, said treatment being characterized in that the subject isadministered on non-consecutive days with two or more doses of said MLVencapsulated cytokine, a first dose of said MLV encapsulated cytokinebeing administered at least 3 days following administration of saidliposome encapsulated chemotherapeutic drug, wherein the time betweenadministration of said MLV encapsulated chemotherapeutic drug and saidliposome encapsulated cytokine is such that the combined therapeuticeffect of said administrations is greater than a sum of the therapeuticeffect produced by administration of the liposome encapsulatedchemotherapeutic drug alone by administration of said MLV encapsulatedimmunostimulating cytokine alone.
 11. The method of claim 10, whereinsaid cytokine is selected from the group consisting of interleukin-2(IL-2), IL-12, IL-15, IL-18, INF-γ, INF-α, INF-β, G-CSF, and GM-CSF. 12.The method of claim 11, wherein said cytokine is IL-2.
 13. The method ofclaim 10, wherein the MLV encapsulating said immunostimulating cytokinecomprise at least one lipid selected from the group consisting DMPC,DMPG, DSTAP, phosphatidyl choline, phosphatidyl ethanolamine andcholesterol.
 14. The method of claim 13, wherein said cytokine isencapsulated in MLV comprising (1) DMPC and (2) at least one additionallipid selected from the group consisting of DMPG and DSTAP, said atleast one additional lipid being in an amount of up to 50%.
 15. Themethod of claim 14, wherein said MLV comprise of DMPC and DMPG.
 16. Themethod of claim 15, wherein the MLV DMPC and DMPG are present in a molarratio of about 9:1.
 17. The method of claim 10, wherein the liposomesencapsulating said chemotherapeutic drug comprise 1-10 mole percent of alipid having a polar head group dericatized with a polyethylene glycolchain which has a molecular weight of between 750 and 10,000 dalton. 18.The method of claim 10, wherein said chemotherapeutic drug is selectedfrom the group consisting of a chemotherapeutic anthraquinone,cisplatin, and a topoisomerase I inhibitor.
 19. The method of claim 18,wherein said chemotherapeutic drug is doxorubicin (adriamycin).
 20. Themethod of claim 19, wherein said chemotherapeutic drug is polyethyleneglycol-coated liposomal doxorubicin.